Biotechnology Progress最新文献

RETRACTION: H. Faraji, M. Ramezani, B. Mashkani, H. R. Sadeghnia, H. M. Benhangi, S. H. Teshnizi, and F. Soltani, “ Comparison of Expression Optimization of New Derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK,” Biotechnology Progress 35, no. 4 (2019): e2819. 10.1002/btpr.2819.

The above article, published online on 11 April 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between journal Editor-in-Chief, John A. Morgan; American Institute of Chemical Engineers, and Wiley Periodicals, LLC. A third party reported that Figure 7 contained several repeated image elements and that a number of these elements were copied from a previous publication by some of the same authors (Faraji et al. 2017 [https://doi.org/10.1080/10826068.2016.1252924]). An investigation by the publisher confirmed these concerns and also found that the protein marker in Figure 9 had been copied from another publication (Pednekar et al 2016 [https://doi.org/10.3389/fimmu.2016.00567]) and that elements in Figure 11B had been duplicated and manipulated.

The authors did not respond to an inquiry and request for original data by the publisher. The retraction has been agreed to because the evidence of image manipulation fundamentally compromises the editors’ confidence in the results presented.

Nanoemulsions, with droplet sizes between 20 and 200 nm, have emerged as a promising vaccine adjuvant and drug delivery system, enhancing the solubility of hydrophobic drugs for diverse applications. Sterile filtration of nanoemulsions is particularly challenging due to the similar size between the nanodroplets and the 0.2 μm nominal pore size rating of sterile filters. One approach to reducing membrane fouling, and enhancing filtration capacity and yield, is to employ an appropriate prefilter, but there are currently no clear guidelines on how to select the prefilter pore size, chemistry, or morphology for sterile filtration of nanoemulsions. This study examined the performance of a range of prefilters with varying pore morphologies and surface chemistries. Sessile drop contact angles were used to evaluate the prefilter hydrophobicity, and bubble point and mercury intrusion porosimetry were used to evaluate the pore characteristics of the different prefilters. The best performance was achieved using a relatively hydrophobic 0.45 μm prefilter made of polyvinylidene fluoride but modified with a somewhat hydrophilic (oxygen-containing) coating. This prefilter reduced the surface tension of the nanoemulsion and provided more than a 2-fold increase in capacity for a variety of sterile filters. These results provide critical insights into the factors influencing nanoemulsion filtration and offer a framework for selection of appropriate prefilters in biopharmaceutical manufacturing.

Real-time detection of foodborne pathogens such as Staphylococcus aureus (S. aureus) is essential for ensuring food safety. In this study, we evaluate the performance of an electrochemical aptasensor developed from gold nanoparticles (AuNPs)-immobilized screen-printed carbon electrode for the detection of low concentrations of S. aureus in chicken extract media. Using cyclic voltammetry (CV), the dynamic interaction between the aptamer-modified electrode and S. aureus was monitored across four bacterial concentrations of 1, 5, 10, and 20 colony-forming units per milliliter (CFU/mL) at 35-min intervals over 350 min. The aptasensor demonstrated a concentration-dependent response with increasingly lower maximum CV signals and faster time to equilibrium as CFU increased. Real-time kinetic and equilibrium parameters were extracted to understand the binding behavior of the pathogen to the electrode surface. Critical parameters such as the kinetic rate constant (k) of 0.0274 min^-1 and equilibrium dissociation constants ( $K_d$ ) of 7.35 CFU/mL, were derived from the CV signals. Langmuir isotherm modeling yielded a maximum binding capacity ( $B_{\max }$ ) of 33.55 μA. In addition, a Hill coefficient (n_H) of 0.65 was obtained, which indicates a slightly negative cooperativity. These findings demonstrate the capability of the aptasensor for real-time detection of S. aureus, offering a robust framework for field-deployable pathogen monitoring in food matrices.

Various technologies, including precipitation, flocculation, depth filtration, microfiltration, and centrifugation, have been developed to clarify mammalian cell culture fluids. For processing volumes between 2000 and 5000 L, continuous centrifugation followed by depth filtration is the preferred method. This process starts with the removal of cells and large debris through continuous centrifugation, followed by the filtration of small debris and some impurities. The newly introduced single-use centrifuge, designed to prevent cross-contamination and mimic traditional continuous centrifuges, was evaluated for its performance, particularly focusing on its impact on cell lysis and subsequent filtration and purification processes. The single-use centrifuge showed better performance in reducing turbidity and lactate dehydrogenase levels (LDH) in the supernatant, indicating less cell lysis compared to the conventional centrifuge. A separation load factor range of 0.91-2.73 was identified as optimal for balancing centrifugation throughput and product quality. Both centrifuge types had a comparable impact on the performance of subsequent depth filtration, supporting a load capacity of at least 100 L/m². No significant differences in product quality, including SE-HPLC, NR/R CE-SDS, icIEF, HCP, and rDNA, were observed between the conventional and single-use centrifuges. These harvest strategies did not affect the subsequent purification steps. For volumes up to 5000 L, both centrifuge types are viable; however, for larger volumes, the conventional centrifuge is necessary due to the scale limitations of the single-use centrifuge.

Castellano BM, Tang D, Marsters S, Lam C, Liu P, Rose CM, Sandoval W, Ashkenazi A, Snedecor B, Misaghi S. Activation of the PERK branch of the unfolded protein response during production reduces specific productivity in CHO cells via downregulation of PDGFRa and IRE1a signaling. Biotechnol Prog. 2023 Sep-Oct;39(5):e3354. doi: 10.1002/btpr.3354.

We have noticed that in Fig. 4D of the article an immunoblot image representing the actin control was inadvertently depicted again in the BiP panel. We have now updated this figure with the appropriate BiP immunoblot image and have hence corrected the figure accordingly. All the article's conclusions remain unchanged as the article sections relating to the Fig. 4D were originally written based on the corrected figure. Please find the corrected Figure 4D below. This correction notice belongs to Figure 4 legends, section (d), page 7 of the published article:

We apologize for this error.

Biosimilar development of monoclonal antibodies (mAbs) is gaining significant momentum as numerous blockbuster biologics approach their patent expiry in the current decade. A critical challenge in biosimilar development lies in achieving product quality attributes(PQAs) comparable to the innovator product. PQAs in upstream processing are influenced by multiple factors, including cell line selection, media composition, feeding strategy, supplements, and bioreactor process parameters, with physical parameter optimization playing a pivotal role in enhancing both product titer and modulating PQAs. In this study, we systematically evaluated the impact of physical process parameters-pH and temperature along with initial seeding density (ISD)-on N-glycan profiles and charge variants across four biosimilar development projects (Projects 1-4). Stepwise regression models were developed between process parameters and product quality attributes using JMP software to establish parameter-attribute relationships. Our results demonstrated that lowering culture pH reduced %acidic variants and %galactosylation while increasing %basic variants and %afucosylation (AF). Increased culture temperature resulted in an increase in %acidic variants and a decrease in %AF. This parameter-attribute relationships knowledge base was directly applied in experimental design to expedite the development of a fifth mAb biosimilar development (Project 5), substantially reducing experimental iterations and development timelines, exemplifying the practical implementation of Bioprocessing 4.0 principles.

Mammalian cell cultures in bioreactors rely heavily on critical process parameter control to ensure optimal growth, productivity, and reproducibility to produce recombinant therapeutic proteins. Culture pH has been shown to be a critical parameter that influences growth, productivity, and critical quality attributes. Typically, pH is either controlled to a set-point throughout the culture or uses a single pH shift to achieve higher productivity and more desirable charge variant profiles. The pH is usually maintained by CO₂ and base additions. For CO₂ controlled cultures, using a set-point can result in an accumulation of CO₂, which has detrimental effects on mammalian cell growth and protein production. In this study, a dynamic pH profile was implemented that allowed the pH control in the bioreactor to mimic the natural uncontrolled pH profile observed in shake flask cultures. This dynamic pH profile employs multiple pH shifts during the exponential phase of a single IgG₁ producing CHO-K1 cell line. The results show that a dynamic pH profile was able to successfully alleviate CO₂ accumulation and increase the cell-specific, as well as overall culture productivity. Impacts of the dynamic pH profile on product quality attributes, including glycosylation and charge variants, were also evaluated, showing mixed impacts on the glycosylation pattern and a positive impact on charge variants. Since the ideal glycosylation pattern is highly dependent on the intended function of the recombinant antibody, impacts on product quality should be evaluated on a "per process" basis.

The cover image is based on the article Accelerating IND-enabling toxicology studies using protein products from stable pools or pools of clones in Chinese hamster ovary cells by Ying Huang et al., https://doi.org/10.1002/btpr.70040

Microcystins (MCs) are toxins produced by cyanobacteria, posing a significant emerging threat to human and public health. Therefore, control strategies combining frequent toxin monitoring with removal techniques are urgently needed. In this context, microcystin degradation using the bacterial enzyme microcystinase A, originally derived from Sphingosinicella microcystinivorans B9, has been identified as a sustainable and effective approach. To facilitate access to the enzyme, the gene encoding microcystinase A was successfully expressed in the Saccharomyces cerevisiae PE-2 strain. The recombinant microcystinase A was produced as an intracellular enzyme and applied in MC degradation assays. Optimal conditions for enzymatic activity were identified at 42.2°C and pH 6.3. The maximum degradation rate of microcystin was determined to be 3.09 mg/L/h, and a K_m of 2.81 μM was obtained when assays were performed at 37°C and pH 7.4. The recombinant microcystinase A remained fully active for 2 h at 20°C. Exposure to 50°C for 1 h resulted in 60% residual activity, while 30 min at 65°C led to complete inactivation. The enzyme was also denatured when exposed to alkaline pH conditions. Therefore, this study provides key data on recombinant microcystinase A, supporting further investigations into its potential applications for MC degradation, particularly under mildly acidic conditions and temperatures up to 45°C.

Perfusion culture is acknowledged as a promising platform for sustained high-density cell production, while concurrently necessitating stringent control over medium nutrient composition. A multi-component medium optimization strategy has been developed in this study, integrating the targeted feeding approach (TAFE), ¹H nuclear magnetic resonance (¹H NMR) analysis, and definitive screening design (DSD). Nine pivotal amino acids were selected through quantitative profiling of cellular uptake kinetics and literature evidence. Their concentrations were optimized using a DSD within only 24 experimental runs. The optimized formulation was demonstrated to maintain stable cell density, high viability (>97%), and excellent monoclonal antibody production in both shake flask semi-perfusion (38.63 pg/cell/day) and 3L bioreactor systems (45-61.5 pg/cell/day), while significantly reducing the accumulation of lactate and ammonium. These results suggest that the proposed strategy can effectively enhance both productivity and metabolic stability, offering excellent scalability and engineering applicability. This work provides a novel and efficient pathway for the development of perfusion culture media in biopharmaceutical manufacturing.